Vascular calcification is prevalent in patients with diabetes mellitus, which increases their morbidity and mortality. Hyperglycemia is a hallmark of diabetes that leads to adverse vascular complications. Emerging clinical and basic investigations support a strong correlation between hyperglycemia and vascular calcification. However, the precise role of hyperglycemia in regulating vascular calcification and the underlying molecular mechanisms remain unknown. Osteogenic differentiation and calcification of vascular smooth muscle cells (VSMC) directs vascular calcification in the media and intima of diabetic vasculature. Conditions associated with diabetes, including high glucose and oxidative stress, have been reported to induce VSMC calcification in culture. We have found that oxidative stress and high glucose increase the expression of the osteogenic transcription factor Runx2, which is essential and sufficient to induce VSMC calcification in vitro and in vivo. Increased protein modification by O-linked -N-acetyl-glucosamine (O-GlcNAcylation) was observed in diabetic arteries from human and mice, which was associated with increased Runx2 and vascular calcification. O-GlcNAcylation is a dynamic and tightly regulated process, which is as common and ubiquitous as phosphorylation and plays a key role in the regulation of diverse biological processes. O-GlcNAcylation is catalyzed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the transfer and removal of O-GlcNAc on target proteins, respectively. Glucose metabolized via the hexosamine biosynthesis pathway increases the production of UDP-GlcNAc, an active sugar donor for O-GlcNAcylation, which elevates O-GlcNAcylation. Increased O-GlcNAcylation has been associated with the adverse cardiovascular effects of diabetes. Our preliminary results demonstrated that high glucose and oxidative stress increased VSMC O-GlcNAcylation and calcification. Elevation of O-GlcNAcylation via OGA knockdown promoted VSMC calcification. Moreover, deletion of OGT not only decreased high glucose and oxidative stress-induced protein O-GlcNAcylation in VSMC, but also blocked VSMC calcification. Therefore, we hypothesize that inhibition of O-GlcNAcylation by OGT deletion in VSMC decreases vascular calcification in diabetes. Using our newly generated inducible SMC-specific OGT knockout mouse model, we will 1) determine the role of OGT-mediated O-GlcNAcylation in diabetic vascular calcification in vivo; and 2) elucidate O-GlcNAcylation-dependent molecular signals that regulate vascular calcification. These studies will provide important molecular insights into developing new strategies or drugs to prevent or treat vascular calcification in diabetes and other vascular diseases featuring increased O-GlcNAcylation.